The invention relates to gastrointestinal abnormalities.
Gastroschisis is a life-threatening abdominal wall defect that occurs in approximately 1-7 of every 10,000 human births. The defect is thought to originate on the right side of the umbilical cord and may involve the formation of the omphalomesenteric artery. Infants with gastroschisis can be born with abdominal organs outside the body cavity, i.e., protruding through the defect. Factors associated with an increased risk for gastroschisis include a maternal age below 20 years, ingestion of aspirin, and ingestion of pseudoephedrine. The cause of gastroschisis has not been identified.
A novel human gene encoding aortic carboxypeptidase-like polypeptide (ACLP) has been discovered. A mutation in an ACLP gene has now been shown to be associated with the development of gastroschisis. Thus, a mutation in an ACLP gene is indicative of gastroschisis or a predisposition to develop the condition. Accordingly, the invention provides an isolated nucleic acid (e.g., genomic DNA, cDNA, or synthetic DNA) encoding an ACLP. By the term xe2x80x9chuman ACLPxe2x80x9d is meant a polypeptide having the amino acid sequence of a naturally-occurring human ACLP. For example, the invention encompasses an ACLP with the amino acid sequence of SEQ ID NO:2 as well as naturally-occurring variants thereof such as mutant forms associated with gastroschisis or isoforms resulting from alternative splicing of exons of the ACLP gene.
The invention includes a nucleic acid molecule which contains the nucleotide sequence of human ACLP cDNA (SEQ ID NO:1). A nucleic acid molecule which contains nucleotides 140-3613 (ACLP coding sequence), inclusive, of SEQ ID NO:1 or a degenerate variant thereof, is also within the invention. Nucleotides 214-3613 encode an ACLP which lacks the first 25 residues (a putative signal peptide). Preferably, the nucleic acid molecule contains a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 87% identical to the sequence of SEQ ID NO:2. More preferably, the sequence is at least 90% identical to SEQ ID NO:2, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably, the nucleotide sequence encodes a polypeptide the amino acid sequence of which is SEQ ID NO:2.
An isolated nucleic acid molecule containing a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:1, or the complement thereof is also within the invention. The nucleic acid molecule may be a primer useful to amplify ACLP DNA in a polymerase chain reaction (PCR). For example, the nucleic acid is at least 5 nucleotides but less than 50 nucleotides in length. Alternatively, the nucleic acid molecule may encompass the entire coding sequence of ACLP CDNA, i.e., nucleotides 140-3613, inclusive, of SEQ ID NO:1. Preferably, the nucleic acid molecule spans a gastroschisis-associated mutation in an ACLP gene. Such a molecule is useful as a hybridization probe to identify a genetic alteration, e.g., a deletion, duplication, point mutation, or translocation, that indicates that an individual has gastroschisis, is predisposed to developing gastroschisis, or is a heterozygous carrier of a genetic alteration associated with gastroschisis.
By xe2x80x9cisolated nucleic acid moleculexe2x80x9d is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism, flank an ACLP gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a procaryote or eucaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term excludes large segments of genomic DNA, e.g., such as those present in cosmid clones, which contain an ACLP gene flanked by one or more other genes which naturally flank it in a naturally-occurring genome.
Nucleic acid molecules include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Hybridization is carried out using standard techniques such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, (1989). xe2x80x9cHigh stringencyxe2x80x9d refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., hybridization and wash conditions of 65xc2x0 C. at a salt concentration of 0.1xc3x97SSC. xe2x80x9cLowxe2x80x9d to xe2x80x9cmoderatexe2x80x9d stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60xc2x0 C. at a salt concentration of at least 1.0xc3x97SSC. For example, high stringency conditions may include hybridization at 42xc2x0 C. in a solution containing 50% formamide; a first wash at 65xc2x0 C. using a solution of 2xc3x97SSC and 1% SDS; followed by a second wash at 65xc2x0 C. using a solution of 0.1%xc3x97SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an ACLP gene are detected by, for example, hybridization at 42xc2x0 C. in the absence of formamide; a first wash at 2xc2x0 C. in a solution of 6xc3x97SSC and 1% SDS; and a second wash at 50xc2x0 C. in a solution of 6xc3x97SSC and 1% SDS.
The invention also includes a substantially pure human ACLP polypeptide. A substantially pure ACLP polypeptide may be obtained, for example, by extraction from a natural source (e.g., a vascular smooth muscle cell); by expression of a recombinant nucleic acid encoding an ACLP;
or by chemically synthesizing the protein. A polypeptide or protein is substantially pure when it is separated from those contaminants which accompany it in its natural state (proteins and other naturally-occurring organic molecules). Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, ACLP. A substantially pure ACLP may be obtained, for example, by extraction from a natural source (e.g., a vascular smooth muscle cell); by expression of a recombinant nucleic acid encoding an ACLP; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eucaryote but produced in E. coli or another procaryote, or in a eucaryote other than that from which the polypeptide was originally derived.
For expression of recombinant ACLP, an ACLP-encoding nucleic acidc is operably linked to a regulatory sequence, e.g., a promoter. By xe2x80x9cpromoterxe2x80x9d is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible, and may be coupled to other regulatory sequences or xe2x80x9celementsxe2x80x9d which render promoter-dependent gene expression cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 region of the native gene, or within an intron. DNA encoding an ACLP may be operably linked to such regulatory sequences for expression of the polypeptide in procaryotic or eucaryotic cells. By xe2x80x9coperably linkedxe2x80x9d is meant that a coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
To produce recombinant ACLP, a cell containing an ACLP-encoding sequence operably linked to appropriate regulatory sequences is cultured under conditions permitting expression of a nucleic acid molecule. The cell may be a procaryotic cell or a eucaryotic cell. To obtain post-translationally modified, e.g., glycosylated recombinant ACLP, the recombinant polypeptide is produced in a eucaryotic cell, e.g., a yeast or mammalian cell.
An ACLP preferably contains an amino acid sequence that is at least 87% identical to the amino acid sequence of SEQ ID NO:2. More preferably, the amino acid sequence is at least 90% (more preferably at least 95%, more preferably at least 98%, more preferably at least 99%) identical to SEQ ID NO:2. Most preferably, the polypeptide contains the amino acid sequence of SEQ ID NO:2.
The invention also includes polypeptides which contain a portion of naturally-occurring ACLP, e.g., an ACLP fragment containing a lysine-rich/proline rich domain (amino acids 117-164 of SEQ ID NO:2), an ACLP fragment containing a discoidin-like domain (amino acids 385-540 of SEQ ID NO:2), or an ACLP fragment containing a carboxypeptidase-like domain (amino acids 562-969 of SEQ ID NO:2).
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
For polypeptides, the length of the reference polypeptide sequence will generally be at least 10 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 25 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides or 300 nucleotides.
In the case of polypeptide sequences which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Sequence identity can be measured using sequence analysis software (for example, the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters as specified therein.
A substantially pure DNA containing an ACLP promoter/enhancer sequence (SEQ ID NO:3) is useful for directing transcription of DNA encoding all or part of ACLP or of DNA encoding a heterologous polypeptide (e.g., a polypeptide other than ACLP or an ACLP the sequence of which corresponds to a naturally-occurring ACLP of a species other than the species from which the promoter/enhancer sequence is derived). For example, a murine ACLP promoter/enhancer sequence may be operably linked to DNA encoding human ACLP for therapeutic expression of ACLP in human patients. To regulate transcription of the polypeptide-encoding sequence (e.g., developmental stage-specific transcription), the promoter/enhancer sequence is operably linked to a polypeptide-encoding sequence. The ACLP promoter/enhancer sequence directs transcription of a polypeptide-encoding sequence.
By xe2x80x9cpromoter/enhancer sequencexe2x80x9d is meant a DNA sequence located 5xe2x80x2 to the transcriptional start site of the ACLP gene and which contains one or more cis-acting elements which regulate transcription, e.g., cell specific transcription. The elements may be contiguous or separated by DNA not involved in the regulation of transcription, e.g., an enhancer element may be in a position immediately adjacent to the promoter element or up to several kilobases upstream or downstream of the transcriptional start site. The promoter/enhancer DNA is preferably derived from the 5xe2x80x2 region of a mammalian ACLP gene, such as that of the mouse (SEQ ID NO:3), and regulates expression of a polypeptide-encoding DNA to which it is operably linked. The promoter/enhancer sequence regulates developmental stage-specific expression, e.g., expression in embryonic cells, of a polypeptide-encoding sequence.
The invention also includes a vector containing the promoter/enhancer DNA of the invention (operably linked to a polypeptide-encoding DNA sequence), and a vascular smooth muscle cell containing the vector. Also within the invention is a method of directing vascular smooth cell-specific expression of the polypeptide by introducing the vector into a vascular smooth muscle cell and maintaining the cell under conditions which permit expression of the polypeptide, e.g., introducing the vector into a human patient for gene therapy.
A method of detecting a gastroschisis-associated genetic alteration is carried out by providing a sample of DNA or RNA from a patient or fetus, and determining whether the DNA or RNA contains a mutation in a gene encoding an ACLP. Detection of such an ACLP mutation indicates that the patient or fetus has a genetic alteration that is associated with the development of gastroschisis. The presence of a gastroschisis-associated genetic alteration is diagnostic of gastroschisis or a predisposition to developing gastroschisis. The method can also be used to identify heterozygous carriers of a mutation associated with gastroschisis. Such individuals may be asymptomatic but are at risk of having children which are homozygous for an ACLP mutation (and therefore, likely to develop clinical gastroschisis). Tissue samples from adult patients are obtained by conventional means, e.g., biopsy or venipuncture. Prenatal testing is carried out by obtaining fetal tissue samples, e.g., by amniocentesis or chorionic villi sampling.
Patient-derived DNA is examined for genetic abnormalities in the ACLP gene, e.g., by detecting restriction fragment length polymorphisms (RFLPs), deletions, point mutations, or other defects. The diagnostic method includes the step of subjecting the sample to polymerase chain reaction (PCR), using a forward PCR primer complementary to a portion of the antisense strand of the gene, the portion being within (a) a first intron of the gene, or (b) the 5xe2x80x2 untranslated region adjacent to the start codon of the gene; and a reverse PCR primer complementary to a fragment of the sense strand of the gene, this fragment being within (a) a second intron of the gene, or (b) the 3xe2x80x2 untranslated region adjacent to the termination codon of the gene. PCR can also be used to detect mutations in an ACLP promoter or other regulatory sequences using primers that flank the mutation. ACLP mutations and/or aberrant ACLP expression can also be detected using standard hybridization techniques, such as Northern blotting.
Fragments of ACLP are useful to raise ACLP-specific antibodies. Accordingly, the invention includes an antibody, e.g., a polyclonal antisera or a monoclonal antibody preparation, that selectively binds to an ACLP. ACLP-specific antibodies are used to diagnose gastroschisis or a predisposition thereto. For example, a diagnostic method is carried out by providing a tissue sample from a patient or fetus, and detecting expression of an ACLP gene in the tissue sample. Expression is measured by detecting the amount of ACLP-specific antibody that binds to the tissue sample, e.g., by ELISA assay, Western blot assay, or immunohistochemical staining of tissue sections. Expression of ACLP is also measured by detecting the level of ACLP transcript in the tissue sample. Regardless of the method of detection of ACLP expression, a reduction in the amount of expression in the patient-derived tissue sample compared to the level of expression in a normal control tissue sample indicates that the patient or fetus from which the sample was obtained has or is predisposed to developing gastroschisis.
Methods of treating or preventing the development of gastroschisis are also within the invention. For example, one treatment regimen includes the steps of identifying a patient with or at risk of developing gastroschisis, and introducing into cells of the patient an isolated nucleic acid encoding ACLP, e.g., a nucleic acid which contains the nucleotide sequence of 140 to 3613 of SEQ ID NO:1. The cells into which the DNA was introduced produce the recombinant ACLP to compensate for a gastroschisis-associated genetic alteration, e.g., a mutation resulting in reduced production of ACLP or a mutation resulting in the production of a defective ACLP. Rather than administering ACLP-encoding DNA to the patient, an ACLP (e.g., a polypeptide having the sequence of SEQ ID NO:2) or a fragment thereof may be introduced into the patient.
An animal model for gastroschisis is useful to study the development of the condition as well as to evaluate therapeutic approaches to treatment or prevention of gastroschisis. A genetically-altered non-human mammal, all diploid cells of which contain a mutation in an endogenous gene encoding an ACLP, is included in the invention. For example, a mammal with a homozygous null mutation in its ACLP gene(s) develops gastroschisis. Preferably, the mammal is a rodent such as a mouse. The genetically altered non-human mammal produces altered levels of ACLP or mutant forms of ACLP. The levels of ACLP gene product in the genetically altered mammal can be increased or decreased at different time periods during development. By xe2x80x9cgenetically altered mammalxe2x80x9d is meant a mammal in which the genomic DNA sequence has been manipulated in some way. The genetically altered mammal may be a knockout in which the endogenous ACLP sequences have been deleted or otherwise altered to decrease or change the pattern of expression. Alternatively, the genetically altered mammal may be transgenic. For example, the transgenic mammal may express ACLP sequences from another species, may overexpress ACLP gene product, or may express ACLP in tissues and at developmental stages other than those in which ACLP is expressed in a wild type animal.
The nucleated cells of a genetically altered mammal not producing a functional endogenous ACLP may be engineered to encode a human ACLP, and to express functional human ACLP, or, alternatively, ACLP from another heterologous species.
Preferably, the genetically altered non-human mammal is a rodent such as a mouse or a rat, the germ cells and somatic cells of which contain a mutation in DNA encoding ACLP. All diploid cells of such an animal contain a mutation in one or both alleles of the endogenous ACLP gene. The mutation can, for example, be a deletion, an insertion, or a nucleotide substitution. The mutation could be in the ACLP regulatory regions or in the coding sequence. It can, e.g., introduce a stop codon that results in production of a truncated, inactive gene product or it can be a deletion of all or a substantial portion of the coding sequence. For example, one or more exons, e.g., exons 7-15, of an ACLP gene may be deleted. By the term xe2x80x9cnull mutationxe2x80x9d is meant a mutation that reduces the expression or activity level of the protein encoded by the mutated gene by more than 80% relative to the unmutated gene. A mouse harboring such a null mutation is a knockout mouse. An ACLP knockout mouse, i.e., one that harbors a homozygous ACLP null mutation, has been found to have an abdominal defect with an extrusion of abdominal organs i.e., gastroschisis.
The invention also includes a mammalian cell line, e.g., immortalized ACLP deficient cells, the genomic DNA of which contains a null mutation in DNA encoding ACLP. Such cells lack the ability to synthesize full length functional ACLP. The cells harboring the null mutation may be derived from a cell obtained from a ACLP deficient mammal, e.g., an ACLP knockout mouse.
Compounds capable of promoting expression or function of an ACLP may be therapeutically useful to treat gastroschisis. Accordingly, the invention includes a method of screening a candidate compound to identify a compound capable of stimulating expression of an ACLP, e.g., human ACLP, by (a) providing a cell or tissue expressing capable of expressing a ACLP, (b) contacting the cell or tissue with the candidate compound, and (c) determining the amount of expression of the ACLP by the cell. An increase in the amount of ACLP expression in the presence of the candidate compound compared to that in the absence of the candidate compound indicates that the compound stimulates expression of the ACLP.
In addition to diagnostic methods, such as described above, the present invention encompasses methods and compositions for evaluating appropriate treatment, and treatment effectiveness of pathological conditions associated with aberrant expression of ACLP. For example, the ACLP gene can be used as a probe to classify cells in terms of their level of ACLP expression, or as a source of primers for diagnostic PCR analysis in which mutations and allelic variation of ACLP can be detected.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments there and from the claims.